Although neurons are not productively infected with HIV-1, neuronal injury and

Although neurons are not productively infected with HIV-1, neuronal injury and death are frequently seen in the brains of AIDS patients with neurological and neurocognitive disorders. target protein, MEF2, which is implicated in neuronal cell protection. Our results show that expression of pDING in neuronal cells diminishes the level of hyperphosphorylated forms of Cdk5 and MEF2 caused by Tat and the other neurotoxic agents that are secreted by the HIV-1 infected cells. These observations suggest that pDING, through its phosphatase activity, has the ability to manipulate the state of phosphorylation and activity of several factors involved in neuronal cell health in response to HIV-1. plants were incubated in 1 ml of lysis buffer containing 30 mM Tris (pH 7.4.), 167 mM NaCl; 0.1% Nonidet P-40 and protease inhibitors cocktail (Sigma-Aldrich, St. Louis, MO). Cell debris was removed by centrifugation at 10,000 rpm for 5 min and the supernatant was recovered and fractionated through 3, 30, and 50 kDa Millipore Microcon LX 1606 supplier filters (Millipore, Billerica, MA) to separate the 38 kDa pDING from the low molecular weight proteins and other plant organic components. Protein concentration and size fractionation were performed as described previously (Darbinian-Sarkissian et al., 2006). The purity of the pDING protein was determined by SDS-PAGE. Transfection of neuronal cells Human neuronal cells were transfected with expression plasmids (5 g each) using the Lipofectamine 2000 system (Invitrogen) per the manufacturer’s protocol for the transfection of primary cortical neurons Treatment of neuronal cells Human cortical neuronal cells were pre-incubated with pDING (300 ng/ml) for 2 h prior to incubation with rTat 101 (ImmunoDiagnostics, Inc. Woburn, MA), (50 ng/ml) for 56 h, or conditioned media (CM) from HIV-1 infected PBMCs for a total of 56 h. The number of neurons and neuronal processes was measured in the presence of pDING, Tat or both. Infection of peripheral blood mononuclear cells (PBMC) LX 1606 supplier with HIV-1 Approximately LX 1606 supplier 1 106 PBMC were incubated with the JR-FL strain of HIV-1 (50 ng of p24-containing virus stock) with serum-free medium for 2 h at 37 C. PBMC cells were then washed twice with PBS, and fresh medium was added. In parallel, control uninfected cells were prepared six days post-HIV-1-infection. Conditioned media from infected and uninfected PBMC cells were used to treat neuronal cells for 56 hours. Rabbit polyclonal to HS1BP3 Infectivity of the cells with HIV-1 was verified by p24 ELISA assay. Preparation of protein extracts and immunoblot analysis For preparation of whole cell extract, cells were washed with cold phosphate-buffered saline (PBS) and solubilized in lysis buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 0.1% Nonidet P-40 and 1% protease inhibitors cocktail (Sigma). Cell debris was removed by centrifugation for 5 min at 4 C. For Western blot analysis, LX 1606 supplier 50 g of proteins were eluted with Laemmli sample buffer, heated at 95 C for 10 min, separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to supported nitrocellulose membranes as described previously (Darbinian-Sarkissian et al., 2006). Proteins were visualized with the enhanced chemiluminescence detection system, ECL+ according to the manufacturer’s instructions (Amersham Pharmacia) and exposed to X-ray film. Western blot analysis of extracts from pDING and Tat expressing cells was performed for Cdk5 and MEF2. Grb2 served as a loading control. Neuronal cell morphology analysis Neuronal cell complexity and outgrowth were evaluated after three days of transfection or treatment. Neurite outgrowth was analyzed by Sholl method (Braunewell et al., 2011; Yanni and Lindsley 2000). Briefly, the percentages (<50 microns, 50-100 LX 1606 supplier microns, >100 microns), averages and standard deviations are shown. The number of dendritic spines and their distribution in cortical neurons was determined by confocal microscopy and the values were analyzed by Image J software (NIH) (Homma et.